Phosphorylation and dephosphorylation of various amino acid side chains (e.g., serine, threonine, and tyrosine) is a major mechanism for regulating protein function in eukaryotic cells. Protein kinases and phosphatases regulate nearly all aspects of cell physiology and have emerged as therapeutic targets for a variety of human diseases. The persistent challenges in kinase and phosphatase inhibitor drug discovery have been poor selectivity across the family of enzymes and high intracellular substrate concentrations. In recent years, it has been recognized that covalent inhibitors afford a mechanism to overcome these hurdles and exhibit a distinct pharmacology as compared to non-covalent inhibitors (e.g., prolonged pharmacodynamics and potential to overcome resistance mutations).
Conventional irreversible inhibitors of kinases and phosphatases inactivate their target through covalent attachment to a specific cysteine thiol functional group. However, the electrophilic center (e.g., acrylamide, haloacetamide, vinyl sulfonamide) that reacts with the cysteine residue can show nonspecific reactivity toward other cellular thiols, including glutathione that is present at millimolar concentrations inside mammalian cells. The electrophile may also cross-react with other nucleophilic functionalities present in biological systems (amino and imidazole groups of amino acids, various reactive sites in nucleic acid bases, water). As a result, the high concentration of nucleophiles present in a cell can render traditional approaches to irreversible inhibition problematic (e.g., toxicity, potency). In view of the foregoing, an acute need exists to develop new chemical methods for covalent targeting of kinases and phosphatases.